WO2012083099A1 - Biologically-active radiolabeled cry1fa and receptor binding assay methods - Google Patents

Abstract

Cysteine-specific radiolabeled Cry1Fa protein retains insecticidal activity against insect pests and binds to insect brush border membrane vesicle receptors in a saturable manner. The biologically-active radiolabeled Cry1Fa protein is useful in competitive binding assays with other Cry toxins.

Description

Biologically-Active Radiolabeled Cry 1 Fa and Receptor Binding Assay Methods

CROSS-REFERENCE TO RELATED APPLICATION

[00011 This application claims the benefit of U.S. Provisional Application Serial No. 61 /423,844, filed December 16, 2010, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

BACKGROUND OF THE INVENTION

[0002] The general field of the invention is the area of life sciences, particularly agricultural sciences and testing methods. Specifically, the field of the invention is the biochemistry and mode of action of Bacillus thuringiensis crystalline (Cry) endotoxins and their interactions with insect Cry toxin receptors.

[0003] Cry insecticidal proteins are endotoxins produced by Bacillus thuringiensis (Bt), a Gram positive bacterium found globally distributed in different soil types. Various classes of Cry proteins are selectively toxic against certain insect pests. The endotoxin is typically found in the form of a crystalline protein located in large inclusion bodies of the bacterium. Cry toxins have considerable sequence diversity (Crickmore et ah, 1998; de Maagd et ai, 2003), but the majority of the toxins that have activity against lepidopteran pests are 130 kDa protoxins having a three domain active core toxin structure (de Maagd et ai , supra).

[0004] The subject of the current invention relates to the Cry 1 Fa toxin, a three domain Cry protein. This toxin has demonstrated insecticidal activity against various lepidopteran insects, including Spodoptera frugiperda (J. E. Smith) (fall armyworm) and Osirinia nubilalis (Hilbner) (European corn borer), which are two of the most economically important insect pests of maize. Cry 1 Fa is the toxin component of two USDA deregulated transgenic plant incorporated pesticides know as event TCI 507 in maize (HERCULEX^®) and event 281-24- 236 in cotton (WIDESTRIKE^®).

[0005] Cry 1 Fa full-length holotoxin protein, like other three-domain Cry toxins, requires proteolytic cleavage at both the N-terminus and the C-terminus ends for activation of its insecticidal activity. The midguts of lepidopteran insects contain a variety of trypsin and chymotrypsin-like proteases that process the full length holotoxin to a core toxin structure having a size of approximately 68 kDa (Christeller et al., 1992; Gatehouse et ai, 1997; Bernardi et al, 1996). The processing involves removal of approximately 28 amino acids from the N -terminus and approximately 530 amino acids from the C-tcrminus (protoxin segment), and the resulting core toxin segment is released and binds to specific receptors located within the insect gut.

[0006] insects can develop resistance to the activity of Cry protein toxins through changes in midgut localized receptors that bind the Cry protein core toxin (Heckel et al, 2007; Van Rie et al , 1990b). Further, other mechanisms of resistance development have been documented, including: reduced activation of the protoxin, changes in the number of Cry receptors in the insect midgut, and the loss of the ability to respond to the toxin In^¬ formation of membrane pores that contribute to insect mortality (see Griffitts and Aroian, 2005; and Van Rie et al, 1990b).

[0007] Prior to this invention, studies to characterize the binding of Cry 1 Fa core toxin protein to different insect receptors have not been reported. The reason being was that traditional radio-labeling methods involving oxidized iodine isotopes reacting with tyrosine residues in the Cry 1 Fa toxin produced radiolabeled core toxin protein that lost its ability to bind to receptors in brush border membrane vesicles (BBMVs) prepared from midguts of Spodoptera exigua and Spodoptera frugiperda (Luo et al, 1999). Further, it was found that traditionally radiolabeled Cry 1 Fa core toxin protein lost its insecticidal activity and was inactive in diet bioassays against these Spodoptera species. Other methods of protein labeling such as fluorescent labeling, and other methods to measure ligand-receptor binding such as isothermal calorimetry, have been attempted, but have been found to be either too insensitive, or the optical methods too difficult to use due to the particulate properties of insect BBMVs.

[0008] Palmer et al. (1997) described an indirect method of radioactive labeling of proteins specifically at cysteine residues. In this method, an intermediate compound, fluorescein-5-maleimide, is first reacted with radio-iodine, then the radiolabeled fluorescein- 5-maleimide is used to chemically modify the protein at available cysteine residues. This invention describes the use of the highly specific Palmer et al. method to radiolabel Cry 1 Fa protein by targeting a single cysteine residue (C205) located in Domain 1 of the Cry 1 Fa core toxin.

[0009] It was most surprising to find that the introduction of this stearically cumbersome radiolabeled 5-maleimide into the Cry 1 Fa core toxin protein prepared by this method did not result in a loss of binding to its receptor or cause toxin inactivation. As a result, this non- traditionally radiolabeled CrylFa core toxin maintained sufficient tertiary protein structure to retain both its insecticidal activity and its ability to bind specifically to receptors in BBMV preparations from a variety of insects.

[0010] The non-traditionally radiolabeled Cryl Fa protein was found to bind to receptors in a saturable manner, and was used in a competitive binding assay to determine if other Cry toxins compete with its binding. Using this assay it was demonstrated that field resistance to CrylFa toxin that developed in a population of S. frugiperda collected in Puerto Rico is due to the loss of the ability of receptors in BBMVs of these insects to bind CrylFa core toxin protein.

BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1 is a bioassay of trypsin-truncated CrylFa and fluorescein-5-malemide labeled, trypsin-truncated CrylFa against S. frugiperda larvae. The figure summarizes the results of insect diet bioassays with Spodoptera frugiperda neonate larvae using iodinated (nonradioactive) CrylFa core toxin protein prepared by the method of the current invention.

[0012] Figure 2 shows saturation binding curves of radio-iodinated CrylFa core toxin protein to European Corn Borer (ECB) BBMVs. The figure depicts the saturation binding of radioiodinated Cry 1 Fa core toxin protein prepared by the method of the current invention to BBMVs prepared from Oslrinia nubilalis larval midguts.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFICATIONS

[0013] SEQ ID NO: l is the synthetic DNA sequence encoding CrylFa holotoxin.

[0014] SEQ ID NO:2 is the CrylFa holotoxin.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Three structural domains of the CrylFa protein are located within the "tryptic core" that is resistant to further trypsin digestion. Domain I is composed of seven a-helices which are believed to insert into the membrane of the insect midgut and form a pore-like structure (Ballester et al., 1999). Domain II consists of three antiparallel β-sheets, and Domain III forms a β-sandwich structure (Pigott and Ellar, 2007). Exposed regions of domains II and III are believed to interact with specific receptors located on the luminal surface of the insect midgut and bind tightly to these receptors (Bravo et al., 2007; de Maagd et al, , 1996; de Maagd et al., 1999; Gomez et al., 2006; Herrero et al., 2004; Lee et al., 1999). Binding of the toxin to a receptor is a necessary requirement for insecticidal activity, and provides the specificity and selectivity of the toxins (Pigott and Ellar, 2007; Rausell et al, 2004). The specificity of activity of different Cry proteins against different insect species has been shown to be related in part to differences in receptors found in different insects (Gomez et al, 2003; Gomez et al, 2007; Van Rie et al., 1990a).

[0016] Quantitative biochemical assays provide a means to measure the interaction of Cry toxins with receptor proteins found in insect midguts. In saturation type binding assays, the labeled Cry toxin protein (ligand) is incubated in buffer solutions that promote the binding of the Cry toxin protein to an insect receptor protein. In one embodiment of a saturation binding assay, a fixed amount of insect receptor protein (commonly brush border membrane vesicles prepared from insect midguts) is mixed (in separate tubes) with increasing amounts of iodinated Cry toxin protein, and allowed to react for a set amount of time.

Unbound Cry protein (that is, not bound to an insect receptor protein) is separated from bound protein by one of various methods, and the amount of radioactivity in the bound protein fraction provides an indication of the amount of Cry protein bound to the insect receptor. One skilled in the field of biochemistry will realize that an observation of little or no bound radioactivity will indicate the lack of a receptor for the particular Cry toxin being studied in the insect species from which the receptor preparation was made.

[0017] A second embodiment of a binding assay is a competitive assay. In a competitive binding assay, the radioactive Cry toxin protein is mixed with an excess of a second, nonradioactive Cry protein, and the Cry proteins are allowed to bind under standard conditions to insect receptor proteins. If the second, non radioactive Cry toxin protein is able to compete with the radioactive Cry toxin protein for binding to the insect receptor, then the radioactive protein will be displaced from the receptor binding site, and little radioactivity will be recovered in the bound state. If the nonradioactive and the radioactive Cry toxin proteins bind to different insect receptor proteins, then they will not compete with one another in binding to the same insect receptor protein. In this instance, the amount of radioactivity recovered in the bound protein fraction will be the same, or nearly the same, as when the nonradioactive second Cry toxin protein was not present in the binding reaction. Various methods are available to quantify the amount of radioactivity in the bound protein fraction.

[0018] Competitive binding assays using I-Cryl Fa core toxin protein and either nonradioactive Cry 1 Fa core toxin protein (control reaction ) or Cry ] Ab core toxin protein demonstrated that both of the nonradioactive core Cry toxin proteins were able to displace the labeled Cry 1 Fa core toxin from its receptor site in BBMV's from S. frugiperda. By contrast, the competitive binding assay demonstrated that that Cryl Ca core toxin protein did not compete with Cry 1 Fa core toxin binding. The results of these binding assays demonstrate that the insect resistance to Cry 1 Fa toxin observed in field studies in a population of S.

frugiperda collected in Puerto Rico can be explained by the inability of receptors in brush border membrane vesicles from these insects to bind Cry 1 Fa core toxin.

[0019] All patents, patent applications, provisional applications, and publications re I erred to or cited herein are incorporated by reference in their entirety to the extent they arc not inconsistent with the explicit teachings of this specification.

[0020] Nucleic acid sequences are presented in the standard 5' to 3' direction, and protein sequences are presented in the standard amino (N) terminal to carboxy (C) terminal direction.

[0021] Unless specifically indicated or implied, the terms "a", "an", and "the" signify

"at least one" as used herein.

[0022] Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. All temperatures are in degrees Celsius.

EXAMPLE 1

Construction of expression plasmids encoding Cry 1 Fa toxin protein

and expression in bacterial hosts

[0023] Standard cloning methods (as provided by, for example, Sambrook et al., (1989) and Ausubel et al., (1997), and updates thereof) were used in the construction of pDAB1817, a Pseudomonas fluorescens (Pf) expression plasmid engineered to produce a chimeric toxin protein (herein referred to as Cry 1 Fa toxin; SEQ ID NO:2) comprised of a Cry 1 Fa core toxin segment (amino acids 1 to 603) and a CrylAb protoxin segment (amino acids 604 to 1 148), encoded by a plant-optimized coding sequence (CDS; SEQ ID NO: l).

[0024] pDAB 1817 is derived from pMYC1803 (US Patent No. 7338794). The basic cloning strategy entailed ligating a DNA fragment, flanked by Spel d Kpnl restriction enzyme recognition sites and containing the Cry 1 Fa toxin CDS, to the large fragment of pMYC1803 prepared by cleaving pMYC^' 1803 DNA with Spel and Kpnl restriction enzymes. Plasmid pMYC1803 is a medium copy plasmid derived from the RSFlOlO-based plasmid pTJS260 (see US Patent No. 5169760), and carries a regulated tetracycline resistance marker and the replication and mobilization loci from the RSF 1010 plasmid. By this means, the Cry 1 Fa toxin CDS was placed under the expression control of the Ptac promoter and the rrnBTlT2 terminator from plasmid pK 223-3 (PL Pharmacia, Milwaukee, Wl). The expression plasmid pDAB 1 817 was transformed by electroporation into P. fluorescens strain MB217 (a derivative of strain MB 101 ; P. fluorescens biovar I), with selection for resistance to tetracycline. Recombinant colonics were identified by restriction digestion f miniprep plasmid DNA.

100251 Growth and Expression Analysis in Fermentors: Production of Cry 1 Fa toxin protein for biochemical manipulation and insect bioassay was accomplished by fermentor- grown P. fluorescens expression strain isolate DR1649. A seed culture was grown 20 hours (final Optical Density at 600 nm = 14) at 32° in a shake flask containing 600 mL of Ps20 medium supplemented with 15 ng/'mL tetracycline, and was used to inoculate 6.6 L of DGMp2.2 medium with tetracycline in a 20 L fermentor tank (New Brunswick Scientific BioFlo 4500, Edison, NJ). Fermentation was carried out at 32° with agitation at 200 to 1000 rpm. Details of the microbiological manipulations for P. fluorescens are available in Squires el al. (2004), US Patent Application No. 20060008877, US Patent No. 7681799, US Patent Application No. 20080058262, and Huang et al. (2007), incorporated herein by reference. Glycerol was batch- fed periodically at 10 gm/L in response to dissolved oxygen

concentration. Expression of the Cry 1 Fa toxin CDS via the Ptac promoter was induced by addition of isopropyl- -D-l-thiogalactopyranoside (IPTG). Cultures were monitored throughout the post induction fermentation time to determine cell density, the level of target gene expression, and other parameters. For SDS-PAGE (sodium dodecyl sulfate

polyacrylamide gel electrophoresis) and immunoblot analyses, aliquots of 0.1 mL of whole fermentation broth were frozen for subsequent analysis. At the final time point at 48 hours after IPTG induction (Optical Density at 600 nm = 222; final culture volume =13.5 L), cells from 4 L of culture were harvested by centrifugation at 10000 x g for 90 min. The cell pellets were frozen at -20° or -80° for further processing.

[0026] SDS-PAGE Analysis of fermentation samples: Frozen fermentor cell broths (0.1 mL) were diluted in chilled water by 5 fold and 200 μΤ were sonicated in ice for 10 minutes using a Branson 250 Sonifier (Branson Ultrasonics, Danbury CT) using a 1/8 inch diameter micro tip with a constant output of 20 units. The lysates were centrifuged at 14000 rpm for 20 minutes (4°) and the supernatants removed (soluble fraction). The pellets were then resuspended i 200 μL· of phosphate buffered saline (PBS; 1 1 .9 mM Na:l IP0₄. 137 mM NaCl, 2.7 mM KC1, pH7.4; Sigma- Aldrich, St. Louis, MO). Further dilutions of both the soluble and insoluble fractions of an equivalent of up to 20-fold of the original fermentation broth were performed in PBS. These fractions were then mixed 1 : 1 with Lacmmli sample buffer (Bio-Rad Inc., Hercules, CA;) containing 5% β-mercaptoethanol and boiled for 5 minutes prior to loading 10 μΐ. to 20 μΐ, on a Criterion 10% Bis-Tris gel with MOPS buffer (Bio-Rad Inc.). Gels were stained with Simply Blue SafeStain™ (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.

[0027] Immunoblotting: Standard biochemical methods (as provided by, for example, Sambrook et ah, (1989) and Ausubel et ah, (1997), and updates thereof) were used for protein isolations and immunoblot analyses. Samples were prepared and proteins were separated by electrophoresis through NuPAGE 4-12% Bis-Tris gels in MES running buffer according to the manufacturer's suggested protocol for denaturing electrophoresis

(Invitrogen). Proteins were transferred onto nitrocellulose membrane for 80 min. at 30 V in NuPAGE transfer buffer. Blots were blocked for 1 hour at room temperature in 5% milk/PBST (PBS with 0.05% Tween-20) and then probed with primary antibody (specific for Cry 1 Fa core toxin segment) and then secondary antibodies for one hour each at room temperature in blocking solution, with rinsing in between each antibody for 15 minutes in PBST. Development of blots was done using Pierce's ECL Western blotting substrate according the manufacturer's protocol (Thermo Fisher Scientific, Rockford, IL).

EXAMPLE 2

Purification of Cry 1 Fa core toxin protein from

Pseudomonas-produced inclusion bodies

[0028] Inclusion body preparations: Protein inclusion bodies (IB) were prepared from P. fluorescens DR1649 cells that contained insoluble Cry 1 Fa toxin protein, as demonstrated by SDS-PAGE and MALDI-MS (Matrix Assisted Laser Desorption/Ionization Mass

Spectrometry). A MALDI-R (Reflectron) mass spectrometer from Micromass (Milford, MA) was used to measure peptide masses in accordance with the manufacturer's prescribed methods and recommendations. Frozen cell pellets were thawed in a room temperature water bath. The cells were resuspended to 10% w/v in lysis buffer (50 mM Tris HC1, pH7.5; 200 mM NaCl; 5% Glycerol; 2 mM EDTA disodium salt (Ethylenediaminetetraacetic acid); 0.5% Triton X- 100; and 1 mM DTT (dithiothreitol-added just prior to use)). 25 ml. of protease inhibitor cocktail (Sigma- Aldrich) was added for every 100 gm of cell paste treated. The slurry was passed twice through a Microfluidics Micro fluidizer at 12000+ psi (Microfluidics Intern. Corp., Newton, MA). The lysate was centrifuged at 18000 x g at 4° for 30 min, and the supernatant was retained. The inclusion pellet was washed in lysis buffer without protease inhibitor cocktail until no bacterial odor remained (usually 2 or 3 washes), by gently homogenizing using a spatula or mechanical mixer (to about 10%> w/v solids) and

centrifuging as described. The resultant superaatants and final pellet were retained and stored at -20°. Small aliquots of each sample were taken and stored in a microcentrifuge tube for SDS-PAGE analysis. The inclusion body preparation pellets were then freeze-dried using a Virtis Advantage freeze dryer (Viopharma Process Systems LTD, Winchester, UK) at ambient shelf temperature and a maximum vacuum drawn for 2 days. The resulting powders were then stored at -20° until analyzed.

[0029] SDS-PAGE analysis and quantitation of IB preparations: Twenty-five mg of IB pellet were resuspended in 1 mL of HTS buffer (20 mM Tris HCL, pH7.5: 50 mM NaCl; 5% v/v glycerol, 10 mM EDTA disodium salt; 0.5% v/v Triton X-100) and sonicated as above for 1 minute on ice. The resuspended samples were then diluted 1 : 1 in Laemmli sample buffer containing 0.2 M DTT. The samples were then diluted 10X and 20X with Laemmli sample buffer without DTT and 10 μΕ were loaded onto a Criterion 18 well 10% Bis-Tris gel (Bio-Rad Inc.) run with IX NuPAGE MES buffer (Invitrogen). Gels were run for 5 min at 100V and then 45 min at 200V. Gels were rinsed and washed in water for 20 min and stained with Simply Blue SafeStain™. Quantification of target bands was done by comparing densitometric values for the bands against a series of Bovine Serum Albumin (BSA) standard samples run on the same gel, and scanned to generate a densitometric standard curve.

[0030] Truncation and purification of CrylFa core toxin protein: Purified inclusion bodies were washed with sterile water, then solubilized in 20 mM CAPS, pHl 1 (3- (cyclohexamino) 1 -propanesulfonic acid) containing 10 mM DTT by rocking the protein solution for 1 hr at room temperature. After removing insoluble material by centrifugation (30000 x g for 30 min at 4°), trypsin treated with 0.5% (w/v) tosyl phenylalanyl chloromethyl ketone (TPCK) (Sigma- Aldrich) was added to the supernatant. This solution was incubated with mixing for 1 hr at room temperature, filtered and concentrated 5-fold using an Amicon Ultra- 15 regenerated cellulose centrifugal filter device (30000 Molecular Weight Cutoff; Millipore), then loaded onto a Pharmacia Mono Q 1010 column equilibrated with 20 mM CAPS pi 110.5. After washing the loaded column with 2 column volumes of buffer, the truncated toxin protein was eluted using a linear gradient of 0 to 0.5 M NaCl in 20 mM CAPS pH10.5 in 15 column volumes at a flow rate of 1.0 mL/min. Purified trypsin-truncated Cry 1 Fa core toxin eluted at about 0.2 M to 0.3 M NaCl. The purity of the proteins was checked by SDS-PAGE with visualization using Coomassie brilliant blue dye (below). In some cases, the combined fractions of the purified toxin were concentrated and loaded onto a Superose 6 column (1.6 cm dia., 60 cm long; GE Healthcare Life Sciences), and further purified by size exclusion chromatography. Fractions comprising a single peak

corresponding to the monomeric size of the truncated core toxin (ca. 68 kDa) were combined and concentrated, resulting in a preparation that was greater than 95% homogeneous for a protein having a molecular weight of about 68 kDa, as judged by SDS-PAGE.

[0031] SDS-PAGE analysis of purified CrylFa core toxin: SDS-PAGE analysis of trypsin truncated CrylFa toxin protein was conducted under reducing and denaturing conditions by the method of Laemmli (as per Sambrook et al, supra). Reduction of the proteins was achieved using 5% β-mercaptoethanol, and heat denaturation was performed at 90° for 5 minutes in the presence of 2% SDS. Proteins were loaded into wells of a 4% to 20% Tris-glycine polyacrylamide gel (Invitrogen) and separated at 200 volts for 60 minutes. Protein bands were stained with Coomassie Brilliant Blue R-250 (Bio-Rad) for one hour, and then destained with a solution of 5% methanol in 7% acetic acid in the presence of cellulose sponges. The gels were imaged and analyzed using a Bio-Rad Fluro-S Multi Imager™ with Quantity One™ imaging software. Relative molecular weights of the protein bands were determined by including a sample of BenchMark™ Protein Ladder (Life Technologies, Rockville, MD) or See Blue™ prestained molecular weight marker (Invitrogen) in one well of the gel.

EXAMPLE 3

Preparation of specifically radio-iodinated CrylFa core toxin protein

[0032] Iodination of CrylFa core toxin protein: Previous work demonstrated that iodination of CrylFa toxin protein destroyed the capacity of the iodinated protein to bind to its receptor(s) in insect brush border membrane vesicles (BBMVs), as well as the insect toxicity of the protein (Luo et ah, 1999). In that study, CrylFa toxin protein was radio- iodinated using a standard iodination bead method (Pierce lodination Beads; Thermo Fisher Scientific), and binding studies revealed that the protein had lost all of its ability for specific binding to receptor(s) in BBMVs from S. exigna and S.fr giperda. Further, when non- radiolabeled Nal was used to iodinate CrylFa toxin protein employing the Iodination Bead method, the iodinated CrylFa was found to have lost its insecticidal activity against

Spodoptera larvae in diet bioassays.

[0033] Iodination of proteins by Iodination Bead protocols takes place at positions ortho to the hydroxy] group on tyrosine; mono- or di-substitution can occur. Thus, the positions of iodination depend on the placement of tyrosine residues within the subject protein, and multiple iodinations may have the consequence of disruption of the protein's structure and/or function. It is noted that the CrylFa core toxin segment comprises 20 tyrosine residues, which may serve as iodination targets.

1 0341 This example teaches an alternative method for labeling the CrylFa core toxin protein with radioactive iodine. Subsequent Examples teach that the radio-labeled core toxin protein binds to insect BBMVs, and that iodinated Cry 1 Fa core toxin protein is active in insect diet bioassays. Further, these Examples teach that trypsin truncated CrylFa core toxin protein can be specifically fluorcscently labeled at the cysteine corresponding to the C205 residue of the Cryl Fa toxin protein using fluorescein-5-maleimide, and that the tluorescently labeled protein is biologically active, causing insect mortality at dosages equal to those of nonlabeled, trypsin truncated CrylFa core toxin.

[0035] The Cry 1 Fa core toxin segment contains three cysteine amino acid residues, at positions 9, 14, and 205. Truncation of the protein by trypsin treatment removes C9 and C14, providing a core toxin segment that retains the cysteine corresponding to the C205 residue. Palmer et al. (1997) demonstrated that the phenyl rings of fluorescein-5-maleimide can be radio-iodinated and then reacted with proteins that contain sulfhydryl groups (e.g. as provided by free cysteine residues), resulting in alkylation of the free cysteines in the protein, and thus providing a radioactively labeled protein. The trypsin-truncated CrylFa core toxin contains a single cysteine residue at the position corresponding to C205, and thus provides a substrate for alkylation and radiolabeling of the protein at a single (specific) site.

[0036] Fluoresccin-5-maleimide (F5-M) was dissolved to 10 raM in DM SO (Dimethyl Sulfoxide), then diluted to 1 mM in phosphate buffered saline (PBS; 20 mM sodium phosphate, 0.15 M NaCl, pH7.5), as determined by the molar extinction coefficient of F 5-M (68,000 ^'Όιη^"1). To a 70 μΤ solution of PBS containing two Pierce Iodination Beads (Thermo Fisher Scientific), 0.5 mCi of Na¹²³! was added behind lead shielding. (An analogous procedure was performed using non-radioactive Nal to prepare (iodinated, non radioactive) fluorescently labeled Cry 1 Fa core toxin protein.) The solution was allowed to mix at room temperature for 5 min, then 10 μΕ of the 1 mM F 5-M solution were added. After reacting for 10 min. the solution was removed from the iodination by pipetting and 2 μg of highly purified trypsin-truncated Cry 1 Fa core toxin protein in PBS were added to the solution. The protein was incubated at 4° with the iodinated F 5-M solution for 48 hrs, when the reaction was terminated by adding β-mercaptoethanol to 14 mM final concentration. The reaction mixture was added to a Zebra™ spin column (Invitrogen) equilibrated in 20 mM CAPS, 150 mM KG, pH9, and centrifuged at 1500 x g for 2 min to separate non-reacted iodinated dye from the protein. The I radiolabeled fluorescein-CrylFa core toxin protein was counted in a gamma counter to determine its specific radioactivity, assuming 80% recovery of the input toxin protein.

[0037] The specific activity of the radiolabeled Cry 1 Fa core toxin protein was approximately 1.1 μθϊ/μ^ protein. This specific activity was low compared to typical expected levels of labeling of similarly-sized proteins using the Iodination Bead procedure. This is presumably due to a single site of labeling (corresponding to C205), and to the (possibly) relative inaccessibility of that site, which is presumed to be located deep within Domain I of the core toxin segment (based upon localization using a comparison crystal structure of the Cryl Aa protein).

[0038] The radiolabeled protein was also characterized by SDS-PAGE and visualized by phosphor-imaging to validate that the radioactivity measured was covalently associated with the Cryl Fa core toxin protein. Radio-purity of the radio-iodinated Cryl Fa core toxin protein (and subsequent detection of Cryl Fa bound to its BBMV receptor) was determined by SDS- PAGE, phosphor-imaging and gamma counting. Coomassie stained SDS-PAGE gels were imaged by wrapping them in Mylar™ film (12 μιη thick), and exposing them under a Molecular Dynamics (Sunnyvale, CA) storage phosphor screen (35 cm x 43 cm) for 1 hour. The plates were developed using a Molecular Dynamics Storm 820 phosphor-imager and the image analyzed using ImageQuant™ software. The radioactive band along with areas immediately above and below the band were cut from the gel using a razor blade and counted in a gamma counter. Radioactivity was detected in the Cryl Fa core toxin protein band and in areas below the band. No radioactivity was detected in the gel region above the Cryl Fa core toxin protein band. Some radioactivity was detectable in the gel region below the Cry 1 Fa core toxin protein band (i.e. fragments smaller than the Cry 1 Fa core toxin protein). These radioactive contaminants likely represent degradation products of the Cry 1 Fa core toxin protein.

[0039] Fluorescently labeled (non-radioactive, iodinated) Cry 1 Fa core toxin protein thus prepared was used in insect diet feeding bioassays to demonstrate that insect toxicity had not been affected, and radio-iodinated (fluorescently labeled) Cry 1 Fa core toxin protein was used in binding studies with BBMV preparations to demonstrate that receptor binding activities had not been affected.

EXAMPLE 4

Insect diet feeding bioassays

[0040] Trypsin- truncated Cry 1 Fa core toxin proteins, either non -radioactive iodine fluorescein-5-maleimide labeled, or unlabeled, were individually tested for their insecticidal activity in a top load diet bioassay against larvae of Spodoptera frugiperda (fall armyworm; FAW) Larvae of F A W were hatched from eggs obtained from a colony maintained by a commercial insectary (Benzon Research Inc., Carlisle, PA).

[00411 The bioassays were conducted in 128-well plastic trays specifically designed for insect bioassays (C-D International, Pitman, NJ). Each well contained 0.5 mL of Multi- species Lepidoptera diet (Southland Products, Lake Village, AR). A 40 xL aliquot of the purified Cry 1 Fa core toxin protein, diluted to various concentrations in 10 mM CAPS, pH10.5, or 40 xh of a control solution, were delivered by pipette onto the diet surface of each well (26.7

Sixteen wells were tested per sample. The negative control was a CAPS buffer solution blank containing no protein. Positive controls included preparations of full length Cry 1 Fa toxin. The treated trays were held in a fume hood until the liquid on the diet surface had evaporated or was absorbed into the diet. Diet concentrations of the Cry 1 F toxin proteins were calculated as the amount (ng) of Cry IF toxin protein per square centimeter of surface area in the well (1.5 cm ).

[0042] Within a few hours of eclosion, individual larvae were picked up with a moistened camel hair brush and deposited on the treated diet, one larva per well. The infested wells were then sealed with adhesive sheets of clear plastic that are vented to allow gas exchange (C-D International. Pitman, NJ). Bioassay trays were held under controlled environmental conditions (28°, about 40% Relative Humidity, 16:8 (Ligh Dark)) for 5 days, after which time the total number of insects exposed to each protein sample, the number of dead insects, and the weight of surviving insects were recorded. Percent mortality and percent growth inhibition were calculated for each treatment. Growth inhibition (GI) was calculated as follows:

GI = [1 - (TWIT/TNIT)/(TWIBC/TNIBC)]

where TWIT is the Total Weight of Insects in the Treatment,

TNIT is the Total Number of Insects in the Treatment

TWIBC is the Total Weight of Insects in the Background Check (Buffer

control), and

TNIBC is the Total Number o Insects in the Background Check (Buffer

control).

The GI₅o was determined to be the concentration of Cry 1 Fa toxin protein in the diet at which the GI value was 50%; i.e. the growth of the insects on Cry 1 Fa-containing diet was only half that of the insects on diet with no CrylFa protein, as calculated above. The LC50 (50% Lethal Concentration; calculated as the concentration of CrylFa toxin protein in the diet at which 50% of test insects were killed) was not determined, as very little mortality was observed during the 5 day experimental period. Statistical analysis (One-way ANOVA) is done using JMP software (SAS, Gary, NC).

[0043] The labeled and non-labeled CrylFa core toxin proteins were approximately equally active at inhibiting growth of S. frugiperda larvae, causing fifty percent growth inhibition at concentrations between 33 ng/cm² and 100 ng/cni² (Figure 1). This result shows that labeling of trypsin truncated CrylFa core toxin protein with iodinated fluorescein-5- maleimide does not result in loss of insecticidal activity.

EXAMPLE 5

Preparation of brush border membrane vesicles (BBMVs)

[0044] Preparation of Solubilized BBMVs: Last instar larvae of S. frugiperda and Ostrinia nubilalis (European corn borer) were held without food overnight and then dissected after chilling on ice for 15 minutes. The midgut tissue was removed from the body cavity, leaving behind the hindgut attached to the integument. The midgut was placed in 9X volume of ice cold homogenization buffer (17 mM Tris base, pH7.5, 300 mM mannitol. and 5 mM EGTA (Ethylene glycol tetraacetic acid)), supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich P-2714) diluted as recommended by the supplier. The final concentration of cocktail components (in μΜ) were AEBSF (500), EDTA (250), Bestatin (32), E-64 (0.35), Leupeptin (0.25), and Aprotinin (0.075). The tissue was homogenized with 15 strokes of a glass tissue homogenize!-. BBMVs were prepared by the MgCL precipitation method of Wolfersberger (1993). Briefly, an equal volume of a 24 mM MgCl₂ solution was mixed with the midgut homogenate, stirred for 5 minutes and allowed to stand on ice for 20 min. The solution was centrifuged at 2500 x g for 15 min at 4°. The supernatant was saved and the pellet suspended into 0.5X volume of homogenization buffer and centrifuged again. The two supernatants were combined, centrifuged at 27000 x g for 30 min at 4° to form the BBMV fraction. The pellet was suspended into BBMV Storage Buffer (10 mM HEPES pH7.4 (4-(2- hydroxyethy 1)- 1 -pipcrazineethanesul onic acid), 130 mM KG, 10% glycerol) to a

concentration of about 3 mg/mL protein. Protein concentration was determined by using the Bradford method with BSA as the standard (Bradford, 1976). The leucine-p-nitroanilide assay for aminopeptidase N determination was made prior to freezing the samples using a Sigma-Aldrich assay kit following the manufacturer's instructions. The specific activity of this marker enzyme in the BBMV fraction typically increased 7-fold compared to that found in the midgut homogenate fraction. The BBMV preparations were dispensed into 250 μΤ samples, flash frozen in liquid nitrogen, and stored at -80°.

EXAMPLE 6

lodinated CryTFa core toxin protein binding assays to BBMVs

[0045] Binding of ¹²⁵I-CrylFa core toxin protein to BBMVs: To determine the optimal amount of BBMV protein to use in competitive binding assays, a saturation curve was generated. ^l2il radiolabeled Cry 1 Fa core toxin protein (0.5 nM) was incubated for 1 hr at 28° with (). nubilalis (ECB) BBMV proteins in concentrations ranging from 0 to 500 μg/mί. in binding buffer (8 mM NaHPO.,, 2 mM KH₂P0₄, 150 mM NaCl, 0.1% BSA, pH7.4). Total volume was 0.5 mL. Bound I-CrylFa core toxin protein was separated from unbound material by sampling 150 xL of the reaction mixture in triplicate and centrifuging the samples at 14000 x g for 8 min at room temperature. The supernatant was gently removed, and the pellet washed three times with ice cold binding buffer. The bottom of the centrifuge tube containing the pellet was cut off and placed into a 13 x 75 -mm glass culture tube. Each sample was counted for 10 min in a gamma counter. The value of counts-per-minute (CPM) minus background (reactions with no added protein) was plotted versus BBMV protein concentration. The optimal range of BBMV protein to use in each binding reaction was determined to be 100 ng/ml, to 150 μg/mL.

[0046] Binding kinetics: To determine the binding kinetics, a saturation curve was generated. Briefly, 150 μg/mL of ECB BBMV proteins were incubated for 1 hr at 28° with increasing concentrations (ranging from 0.5 nM to 20 nM) of ^l25I-CrylFa core toxin protein. Total binding was determined by sampling 150 μΐ_^ of each concentration in triplicate, followed by cenlrifuging and counting as described above. The triplicate values were averaged. Non-specific binding was determined in the same manner, with the addition of 1 ,000 nM of non-radioactive (competitor) Cry 1 Fa core toxin protein. This concentration was at least 50-fold higher than the highest concentration of the radiolabeled Cry 1 Fa core toxin protein used; thus it would be expected to bind to all available receptor sites, while displacing the radiolabeled Cry 1 Fa core toxin protein. Specific binding was calculated as the difference in CPM (minus background) between total binding and non-specific binding. It was determined (Figure 2) that I-Cry l Fa core toxin protein specifically bound to the ECB BBMV proteins in a saturable manner, with a ¾ of 1 1 nM and a B_max of 37 fmoles/mg of ECB BBMV proteins. The level of non-specific binding was nearly 70% of total binding; however the level of specific binding clearly saturated, showing that the interaction was a receptor-mediated binding response.

EXAMPLE 7

Competition binding assays with BBMVs

1 0471 A "pull-down" type receptor assay was used to determine the amount of competition for binding to receptor sites between radiolabeled Cry 1 Fa core toxin protein and other (non labeled) Cry toxin proteins. In this assay the relative molecular size of the bound ligand with receptor was characterized by separation by SDS-PAGE, and the amount of radiolabeled ligand bound to the BBMV receptor was measured by detection of radioactivity in the gel by phosphor-imaging. Homologous and heterologous competition binding assays were conducted using 100 μg/mL or 150 μg/mL of BBMV protein (from various insect sources) and 0.5 nM or 2.5 nM of I-CrylFa core toxin protein. The competitive ligand (i.e. 1000 nM of non-radiolabeled Cry toxin protein) was added to the reaction mixture at the same time as the radioactive ligand to assure true competitive binding interactions, incubations were carried out for 1 hr at 28°, and the ¹²⁵I-Cryl Fa core toxin protein bound to its BBMV receptor(s) was separated from nonbound protein by centrifuging the reaction mixture as described above. The pellet was washed three times with ice cold binding buffer, then solubilized by adding 25 of 2X Laemmli buffer with 5% β-mercaptoethanol, and rapidly mixing the sample for 10 min at 95°. The sample was centrifuged and duplicate samples were loaded onto a 4% to 20% Tris glycine polyacrylamide gel and separated by SDS-PAGE. The amount of ¹²³I-CrylFa core toxin protein bound to receptor was measured by phosphor-imaging of the gel and densitometry of the bands corresponding to radioactive Cryl Fa core toxin protein after a 3 day exposure of the plate.

[0048] In a control reaction, 0.5 nM of ^~ I-CrylFa core toxin protein was incubated with S. frugiperda (FAW) BBMV proteins for 1 hr, and the amount of ¹²⁵I-CrylFa protein that bound to the BBMVs was measured by SDS-PAGE and phosphor-imaging as above. A concurrent experiment, to which was added 1000 nM of unlabeled Cryl Fa core toxin protein, demonstrated that this addition of a 2000-fold excess concentration of unlabeled Cryl Fa core toxin protein completely eliminated the binding of the radiolabeled Cry 1 Fa protein. These results demonstrated that this assay effectively measures the ability of a Cry toxin protein to displace the binding of Cryl Fa core toxin protein to its FAW BBMV receptor(s). In a similar manner, it was determined that 1000 nM of CrylAb core toxin protein completely eliminated the binding of ^12"I-Cryl Fa core toxin protein to FAW BBMV proteins. This result is consistent with other reports indicating that the Cry 1 Ab toxin protein and Cryl Fa toxin protein share similar receptor binding sites (Banks et al., 2001; Hernandez and Ferre, 2005).

[00491 Further, it was found that there was no competition for binding to FAW BBMV receptors between 1000 nM of CrylCa core toxin protein and 0.5 nM of ¹²⁵I-Cryl Fa core toxin protein, indicating that the CrylCa protein binds to a receptor different from the Cryl Fa receptor(s). This result is consistent with other studies conducted with Diatraea saccharalis (sugarcane borer) BBMVs, in which it was found that excess Cryl Fa protein did not displace biotinylated CrylCa protein from its receptor(s).

EXAMPLE 8

Cryl Fa core toxin binding to BBMVs prepared from

FAW insects resistant to Cryl Fa toxin [00501 Pull-down competitive binding assays were performed using BBMVs prepared from insect larvae that are resistant to intoxication by ingested Cry 1 Fa toxin. Cry 1 Fa- resistant larvae of S. frugiperda (rFAW)) were hatched from eggs harvested from a proprietary colony (Dow AgroSciences LLC, Indianapolis, FN). The rFAW larvae are more than 30-fold resistant to the toxicity of Cry 1 Fa toxin. BBMVs were prepared from rFAW larvae in the same manner as from their Cry 1 Fa-sensitive counterparts.

[0051 ] As shown above, BBMVs from wild type (i.e. Cry 1 Fa-sensitive) FAW larvae bound ¹²⁵I-CrylFa, and the binding could be displaced by 2000-fold addition of non-labeled Cryl Fa protein. In contrast, BBMVs prepared from rFAW larvae and used in pull-down binding assays at 1 0 .ug/mL with 2.5 nM ^12"l-Cryl Fa did not bind any of the ¹²⁵l-CrylFa core toxin protein. These results indicate that the resistance to Cryl Fa toxin in rFAW may be due to the inability of the Cryl Fa receptor(s) in the BBMVs to bind Cryl Fa core toxin protein. Alternatively, the resistance may be a result of absence of the receptors from the insects. In any case, it appears that the mechanism of resistance to Cryl Fa toxin in these rFAW larvae is receptor based.

EXAMPLE 9

Cryl Fa core toxin binding to BBMVs prepared

from ECB insects resistant to Cryl Fa toxin

[0052] Pull-down competitive binding assays were performed using BBMVs prepared from insect larvae that are resistant to intoxication by ingested Cryl Fa toxin. Cryl Fa- resistant larvae of O. nubilalis (rECB) were hatched from eggs harvested from a proprietary colony (Dow AgroSciences LLC, Indianapolis, IN). The rECB larvae are more than 30-fold resistant to the toxicity of Cryl Fa toxin. BBMVs were prepared from rECB larvae in the same manner as from their Cryl Fa-sensitive counterparts.

[0053] Pull-down experiments were conducted using 100 ng/mL of BBMVs prepared from wild type ECB larvae {i.e. Cryl Fa-sensitive) and Cryl Fa-resistant (rECB) larvae, and using 2.5 nM of I-Cry 1 Fa core toxin protein. The radioiodinated protein was bound to BBMVs from ECB larvae, but to a lesser extent than was seen for binding to BBMVs from FAW larvae. Non-radiolabeled (competitor) Cryl Fa protein competed with the binding of ¹²⁵I-CrylFa to ECB BBMVs but the competition was less than that which was measured in competition experiments using FAW BBMVs. [0054] Some binding of ¹²⁵I-Cry lFa to rECB BBMVs was detected, and the amount of binding was less than that measured using ECB BBMVs. Non-radiolabcled (competitor) Cry 1 Fa protein did not completely compete with ¹²⁵I-CrylFa core toxin protein for binding to rECB BBMVs. These data support earlier Cry 1 Fa binding studies done by Surface Plasmon Resonance using BBMVs from ECB and rECB. Those studies showed that non-iodinated Cry 1 Fa toxin protein could bind to BBMV fractions prepared from either ECB or rECB larvae, but dissociation of the toxin from the ECB BBMVs was slower than its dissociation from rECB BBMVs. The ¹²⁵l-CrylFa core toxin protein binding detected in the pull-down type assays described here are not sufficiently quantitative to measure binding affinity, but do indicate that Cry 1 Fa toxin protein binds to rECB BBMVs to a significant degree. Thus, resistance to Cry 1 Fa toxin in rECB may be due to less efficient binding of Cry 1 Fa core toxin protein to rECB BBMV receptor (s), as compared to binding to receptors of susceptible larvae. This could manifest as less efficacious for membrane pore formation, since the time that the toxin remains on the receptor is shorter than in susceptible insects.

[0055] Table 1 summarizes the relative binding results from assays using radio-iodinated Cry 1 Fa core toxin protein with BBMV preparations from various insect sources. Percentages indicate the relative amounts of radioactivity bound to BBMVs, with, and without, nonradioactive competitor ligand. 100% values do not represent equal numbers; ECB BBMVs bind less total Cry 1 Fa protein than do FAW BBMVs.

* 1000 nM of nonradioactive competitor Cry core toxin proteins added where appropriate

**N/A = Not Applicable, since very little ¹²⁵I-CrylFa core toxin protein was bound

[0056] This invention demonstrates that, surprisingly, CrylFa core toxin protein can be radio-labeled using ¹²⁵¹ labeled fluorescein-5-maleimide to specifically alkylate cysteine residues while maintaining insecticidal activity and receptor binding capability. The trypsin truncated form of Cryl Fa core toxin contains only a single cysteine residue (corresponding to C205 within Domain 1 of CrylFa), which must comprise the site of radiolabeling. This region constitutes part of the pore structure of the protein and is not thought to be involved in receptor binding. One skilled in the field of protein biochemistry and structure will realize that molecules similar to fluorescein-5-maleimide, which contain a phenol ring attached to a maleimide function, could be iodinated by chloramine chemistry and could be used to iodinate specific cysteine residues in proteins such as CrylFa. In particular, analogs smaller than fluorescein-5 -maleimide might be more accessible to the interior of CrylFa core toxin thus may result in greater specific radioactivity of the labeled protein.

[0057] The cysteine alkylated CrylFa protein produced herein is insecticidally active against FAW larvae. Unlabeled CrylFa protein and unlabeled Cryl Ab protein, but not unlabeled CrylCa protein, can displace the binding of radiolabeled CrylFa protein from FAW BBMVs. Bioassay data demonstrates that CrylCa is active against both FAW and rFAW larvae, while Cryl Ab is less active against rFAW larvae. Thus, the biological activities of CrylFa, CrylAb, and CrylCa proteins against FAW and rFAW larvae are conveniently explained and predicted by the BBMV binding assay results. It is therefore one aspect of this invention that combinations of Cry proteins useful in Insect Management strategies may be predicted using competitive binding assays employing CrylFa protein radiolabeled by means of the methods of this invention.

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Claims

CLAIMS We claim:

1. A protein selected from the group consisting of Cry 1 Fa holotoxin and Cry 11· a core toxin wherein said protein is labeled with a radioactive isotope of iodine and wherein said labeled protein retains the ability to selectively bind to receptors in brush border membrane vesicles prepared from midguts of Spodoptera exigua and Spodoptera frugiperda.

2. The protein of Claim 1 that is Cry 1 Fa holotoxin.

3. The protein of Claim 1 that is Cry 1 Fa core toxin.

4. A saturation-type binding assay method for determining binding activity of Cry 1 Fa core toxin to receptors on the midgut of a given insect species which comprises: a) preparing a suspension containing a known concentration of brush border membrane vesicles isolated from midguts of the insect species;

b) adding, in separate containers, increasing amounts of the labeled Cry 1 Fa of Claim 3 to fixed amounts of the brush border membrane vesicle suspension under time and buffer conditions that permit the labeled Cry 1 Fa to bind to receptors on the brush border membrane vesicles;

c) separating the unbound labeled Cry 1 Fa from labeled Cry 1 Fa bound to the receptors on the brush border membrane vesicles; and

d) counting any radioactive signal emitted from the labeled Cry 1 Fa bound to the brush border membrane vesicles.

5. A competitive binding assay method for determining cross-reactivity which

comprises:

a) preparing a suspension containing brush border membrane vesicles isolated from midguts of an insect species;

b) mixing an the labeled Cry 1 Fa of Claim 3 with an excess of a different unlabeled protein and the brush boarder membrane suspension under time and buffer conditions that permit the labeled Cry 1 Fa and unlabeled protein to bind to receptors on the brush border membrane vesicles;

c) isolating the bound brush border membrane vesicles from unbound labeled Cry 1 Fa and unbound unlabeled protein mixture; and

d) comparing the radioactivity emitted from the bound brush border membrane vesicle fraction to the radioactivity emitted from the unbound mixture fraction.

6. The method of Claim 5 wherein the unlabeled protein is a Cry protein.